An early nodulin cDNA, dd23b, was isolated from white clover root tissue by differential display RTPCR. Its full-length sequence of 340 nucleotides encodes a predicted 72-amino-acid protein of molecular mass 8.3 kDa, with a polypeptide region containing cysteine pairs spaced in the manner of a cysteine cluster protein. This feature, which is shared by some other late and early nodulins from pea and broad bean, suggests a role in metal ion binding and membrane transport. Temporal and spatial expression patterns were determined during infection and nodulation by the homologous microsymbiont. No expression was found in unchallenged root tissue over a 7-day sampling period. Expression was first detectable in roots by RT-PCR 6 h post-inoculation with Rhizobium leguminosarum biovar trifolii, placing dd23b among the earliest nodulins to be detected to date. In root nodules, expression occurred primarily in the central symbiotic zone, but also in some host cells within the infection zone. Addition of purified wildtype chitolipooligosaccharide Nod factor to axenic white clover roots induced dd23b expression, providing further evidence for the role of this gene in the early plant response to infection by rhizobia.